ROS-derived lipid peroxidation is prevented in barley leaves during senescence

Abstract

Senescence in plants enables resource recycling from senescent leaves to sink organs. Under stress, increased production of reactive oxygen species (ROS) and associated signalling activates senescence. However, senescence is not always associated with stress since it has a prominent role in plant development, in which the role of ROS signalling is less clear. To address this, we investigated lipid metabolism and patterns of lipid peroxidation related to signalling during sequential senescence in first-emerging barley leaves grown under natural light conditions. Leaf fatty acid compositions were dominated by linolenic acid (75% of total), the major polyunsaturated fatty acid (PUFA) in galactolipids of thylakoid membranes, known to be highly sensitive to peroxidation. Lipid catabolism during senescence, including increased lipoxygenase activity, led to decreased levels of PUFA and increased levels of short-chain saturated fatty acids. When normalised to leaf area, only concentrations of hexanal, a product from the 13-lipoxygenase pathway, increased early upon senescence, whereas reactive electrophile species (RES) from ROS-associated lipid peroxidation, such as 4-hydroxynonenal, 4-hydroxyhexenal and acrolein, as well as β-cyclocitral derived from oxidation of β-carotene, decreased. However, relative to total chlorophyll, amounts of most RES increased at late-senescence stages, alongside increased levels of α-tocopherol, zeaxanthin and non-photochemical quenching, an energy dissipative pathway that prevents ROS production. Overall, our results indicate that lipid peroxidation derived from enzymatic oxidation occurs early during senescence in first barley leaves, while ROS-derived lipid peroxidation associates weaker with senescence.

FIGURE 1

Changes in photosynthesis‐related parameters during sequential senescence in barley leaves of cv. Lomerit (closed symbols) and cv. Carina (open symbols). (A) Maximum quantum yields of photosystem II (F _v/F _m; circles) and maximum redox change of photosystem I (P _m; squares) of first‐emerging leaves after 13, 20 and 27 days (first time course) or 18, 25 and 32 days (second time course) after sowing seeds, with all changes shown relative to values of pre‐senescent leaves (13 days), means ± sd, n = 6 for each genotype at each time point. (B, C) Relationship of P _m values with (B) F _v/F _m, and (C) total chlorophyll (a + b) concentrations, with each data point representing an individual leaf

FIGURE 2

Relationship between maximum redox change of photosystem I (P _m), used as a senescence marker, and xanthophyll cycle de‐epoxidation ratios (VAZ de‐epox.), and changes in non‐photochemical quenching (NPQ), during sequential senescence of first‐emerging barley leaves of cv. Lomerit (closed symbols) and cv. Carina (open symbols). (A) Each data point represents an individual leaf, V: Violaxanthin; A, Antheraxanthin; Z, zeaxanthin. (B) NPQ values of leaves after 13, 20 and 27 days after sowing seeds, means of each time point ±sd, with different letters denoting significant differences at p < 0.05, n = 6 leaves for each genotype at each time point

FIGURE 3

Concentrations of aldehydes and RES, as markers of lipid peroxidation, during sequential senescence of first‐emerging barley leaves. Differences for cv. Carina (C) and cv. Lomerit (L), and for each time after sowing seeds (indicated left), are shown relative to pre‐senescent leaves (average of both cultivars 13 days after sowing seeds), as log₂ values on a colour scale (shown below). Data have been normalised to (A) total chlorophyll, and (B) measured leaf area, with * and ** denoting significant differences at p < 0.05 and p < 0.01, respectively, n = 6 leaves for each cultivar at each time point. (C) Leaf lipoxygenase activity in cv. Carina relative to maximum redox change of photosystem I (P _m), used as a senescence marker

FIGURE 4

Relationship between maximum redox change of photosystem I (P _m), used as a senescence marker, and tocopherol concentrations during sequential senescence in first‐emerging barley leaves. (A) α‐tocopherol (circles) and γ‐tocopherol (squares) normalised to total chlorophyll in leaves of cv. Lomerit (closed symbols) and cv. Carina (open symbols). Each data point represents an individual leaf with all data shown from both time courses. (B) Concentrations of α‐tocopherol normalised to leaf area in cv. Carina and cv. Lomerit

FIGURE 5

Changes in fatty acids (FA) and membrane lipids during sequential senescence in first‐emerging barley leaves of cv. Carina. (A) Percentage of each FA in total leaf FAs, in pre‐senescent leaves 18 days (d) after sowing seeds. Species are denoted by acyl chain length followed by number of double bonds; linolenic acid (C18:3), palmitic acid (C16:0), linoleic acid (C18:2), stearic acid (C18:0) and palmitoleic acid (C16:1). (B) Fold difference of each fatty acid between 18 d and 25 d (blue), and 25 d and 32 d (yellow) on a log₂ scale. Asterisks denote a significant change compared to the previous time period (p < 0.05). (C) Relative difference of membrane lipids between 18 d and 32 d (n = 4), as quantified by band intensity after separation of total lipid extracts by thin layer chromatography, as shown in (D). All data are normalised to leaf DW.

FIGURE 6

A bi‐plot principal component analysis (PCA) of photosynthetic and lipid peroxide‐related parameters during developmental senescence of first‐emerging barley leaves. Each data point, separated in PC1 (bottom: x‐axis) and PC2 (left: y‐axis), represents an individual mid‐leaf segment of cv. Lomerit (L) and cv. Carina (C), coloured by age, into Groups 1 (13 days since sowing; purple), 2 (18 or 20 days since sowing; red), 3 (25 or 27 days since sowing; yellow) or 4 (32 days since sowing; green). Overlaid is the loading plot (right: y‐axis; top: x‐axis), showing established senescence indicators (black dotted line), photosynthetic pigments and tocopherols (blue), and all detected lipid peroxide breakdown products (grey).